High efficiency ripa lysis buffer

Total proteins in spleens or aortas were extracted via High Efficiency RIPA Lysis Buffer (KeyGEN BioTECH), quantified by BCA protein concentration assay kit (Beyotime Biotechnology), and denatured in boiling water with SDS-PAGE protein loading buffer (Beyotime Biotechnology). 30 μg of proteins per sample was loaded in 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as blocking agent before antibody incubation. Polyvinylidene fluoride (PVDF) membranes were incubated with rabbit antibodies against p-STAT3-Y705 (Abcam), p-STAT5-Y694 (Cell Signaling Technology), SOCS3 (Abcam), FOXP3 (Abcam), β-ACTIN (Abways), and Goat anti-rabbit IgG with HRP (Biosharp), respectively. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Blot data were analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas are measured, and the relative expression amount of the protein samples is calculated by the method of the target protein gray value/internal reference β-ACTIN gray value.

Total protein was extracted from spleens or aortas by High Efficiency RIPA Lysis Buffer (KeyGEN BioTECH) and quantified by BCA protein concentration assay kit (Beyotime Biotechnology). Protein loading buffer (Beyotime Biotechnology) was added into protein and boiled for 5 min for protein degeneration. Thirty microgram of protein was loaded in 8% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as a blocking agent before antibody incubation. PVDF membranes were incubated with rabbit antibodies against Dnmt3b (Cell Signaling Technology) and GAPDH (Proteintech), mice antibody Dnmt1 (Abcam). Goat anti-rabbit IgG and goat anti-mice with HRP (Proteintech) were chosen as the secondary antibody. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Data was analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas were measured, and the relative expression amount of the protein samples was calculated by the method of the target protein gray value/internal reference GAPDH gray value.